JPH07509602A - Novel malignant cell type internal nuclear matrix marker - Google Patents

Abstract

Disclosed are genetic sequences and their encoded amino acid sequences for two interior nuclear matrix proteins useful as markers of malignant cell types. Primary and secondary structure analysis of the proteins is presented as well as means for their recombinant production, and compositions and methods for the use of these markers in clinical assays and cancer therapies.

Description

[Detailed description of the invention] Novel malignant cell type internal nuclear matrix marker Mitsuri Rinki 1 All eukaryotic cells (both animals and animals) have a cough that is surrounded by cytoplasm.

The nucleus forms a complex with proteins and contains cellular DNA called chromatin. nine Lomatin, together with its associated proteins, constitutes a major part of the entire cough, and here It is organized by an internal fluorescent skeleton of the nucleus called Rix (NM).

The nuclear matrix is also digested with DNase I and treated with high concentrations of It is defined as the nuclear structure that remains after removal of chromatin by salt extraction. this bone The lattice nucleus structure is further divided into the “internal nuclear matrix°” (INM) and the nuclear pore plate boundary complex. Characterized by the body.

Various studies have shown that NM is essential for multiple pile functions important for the regulation of gene expression. (For an overview see e.g. Fey et al. 1991) Crit, Rev, Euk, Gene Express, 1: 127443). In particular, U.S. Pat. As described, certain nuclear matrix proteins (particularly the inner nuclear matrix pallidum) white) is useful as a marker protein for identifying cell types. For example, specific IMN! The presence and abundance of white is characteristic of a particular cell type and may be present in a sample. It can be used to identify the tissue of origin of existing cells or cell fragments. Book A particularly important application of the discovery is the use of the marker IN-i protein in the determination of metastatic tissue. be. Expression of certain INM proteins is also associated with malignant or otherwise dysfunctional disease. It is known to change in cells. Also, malignant and/or dysfunctional cells Changes in the expression pattern of these fluorophores are important for diagnostic purposes and tissue viability evaluation. In order to Make it useful as car protein. U.S. Patent Nos. 4,882,268 and 4,885 No. 236 (patent mills are 11/21/89 (Penman) and 121, respectively) 5/89 (Fey)) extracts insoluble INM protein from cells or cell remnants. selectively extracting its associated nucleic acids and revealing this protein in two-dimensional electrophoresis gels discloses a method to identify the expression patterns of these fluorophores in specific cell types by are doing. Additionally, INM proteins or protein fragments can also be extracted from dead cells in soluble form. It was recently discovered that it can be liberated in the form (U.S. Patent Application No. 78580 No. 4, filed on October 31, 1991).

To date, the molecular properties of specific proteins in NM, particularly INM, have not been fully determined. Most obvious by their low abundance and generally insolubility. It wasn't covered.

Isolate specific nuclear matrix proteins and the gene sequences that encode them; It is now possible to determine the properties of these proteins and their nucleic acids. – furthering their use as molecules and further exploring the biological roles of these proteins in vivo. It is expected that this will facilitate the elucidation of

The object of the present invention is to obtain a gene encoding an INM defect that is useful as a marker for malignant cell types. The purpose is to provide the gene sequence. Another objective is to remove these fluorophores in the sample and of nucleic acids (including RNA transcripts). Book Another object of the invention is to provide compositions for use in diagnostic and other histology procedures. Is Rukoto. Yet another objective is to develop genes that are useful as target molecules in cancer therapy. The purpose is to provide the gene sequence and amino acid sequence. These and other of the invention Objects and features will become apparent from the following description, drawings, and claims.

Miyonghui Hill The molecular property data including the DNA sequence data of the two types of INM proteins were Herein, MTI and This protein, called MT2, is present at increased levels in malignant &l1vIi and extracellular fluids. Exists. Therefore, this protein and the gene sequences encoding them are as a marker molecule to identify tissue tumorigenesis in samples or body fluid samples. It is considered to be useful.

Isolate complete or partial clones of genes encoding these proteins, and The DNA sequence, reading frame, and amino acid sequence encoded by these DNAs were determined. The DNA sequence of 0MT2 was determined by Yang et al. (1992) J, Ce1l. Biol, ', 11 Rainbow 1303-1317) and Compton et al. ton et al., (1992) J, Ce1l Biol, Ui-1395-1 408) (referred to as NuMA in those documents). Ru. This nucleic acid (and encoded amino acid sequence), referred to herein as MTI, is , has not been previously reported and has little sequence homology to sequences known in the art. This is a new array. Furthermore, MTI was subcloned in the expression hector. , and further expressed as a fusion protein that can be cleaved in Escherichia coli (L). Immunofluorescence method Both MTI and MT2 (NuMA) are non-mitotic cell Distributed throughout the nucleus of the vacuole (excluding the kernel) and localized to the spindle during mitosis.

The gene sequences described herein include MTI and M72N white (MTI and MT2 (including allelic variants and mutants). this Strain proteins are derived from recombinant DNA, the DNA itself, and these produced by expression in a host cell that has a nucleic acid and is capable of expressing it Contains protein. Recombinantly produced proteins can be prepared using standard methods, e.g. It is possible to separate the proteins using nitty chromatography to obtain substantially pure proteins. As used herein, "substantially pure" means This is understood to mean that it does not substantially contain any contaminant proteins.

The family of proteins defined as MTI is based on the nucleic acid sequence of SEQ ID NO: 1 (including analogs thereof) As used herein, "analog" ” refers to allelic variants and variants, as well as other natural and artificial variants. is understood to include. These variants contain biologically active and inactive forms of this protein. Particularly contemplated, including, are DNAs having a variety of preferred codon usages. , DNA with a “silent mutation” of the DNA of SEQ ID NO: 1 (in this case, the gene Sequence changes do not affect the encoded amino acids), and Deoff et al. ayoff et al., r l tr” J (Atlas of Protein 5equence and 5structure) Volume 5, Appendix 3.345-3 62 (M, O, DayoffW, Nat'l Biolwed, Re5ea rch Foundation+Washington D.C., 1979). This is DNA that encodes conservative amino acid changes.

Therefore, nucleic acids encoding proteins of the ~iTl family are subject to severe hybridization. It can be defined as a sequence that hybridizes with the DNA sequence of SEQ ID NO: l under reaction conditions. . As used herein, stringent hybridization conditions are Molecular Cloning: Laboratory Manual, Maniac-Nice Edition, Cold Spring For example, 5 0% formamide, 5X Denhardt's solution, 5X SSPE, 0.1% SDS and Hybridization was performed in 100 μg/m + denatured salmon sperm and 2XSSC Wash at 37°C with 10°1% SDS, then wash at 37°C with 10. The conditions were washing at °C.

The protein of the family defined as MT2 has the nucleic acid sequence of SEQ ID NO: 3 (including analogs thereof) Contains fluorescein, coded by This analog includes allelic variants and mutations. species, as well as other natural and artificial variants. These variants have biological activity and and inactive protein forms. Particularly envisaged are DNs with silent mutations. A, with DNA encoding other preferred codon usage and conservative amino acid changes. Nucleic acids encoding the MT2 protein lineage of the present invention are subject to severe hybridization. A DNA sequence that hybridizes with the DNA sequence of SEQ ID NO: 3 under the conditions of Wear.

In another aspect, the invention hybridizes with a gene sequence encoding MTI. Nucleic acid fragments (but not necessarily encode functional proteins) ``oligonucleotide'' or ``oligomer''). Tide has converted the gene sequence encoding the protein contained in the ~fT1 protein into cDNA or or from a genomic DNA library, and/or A program for distinguishing between the white-encoding sequence and the naturally associated gene sequence. lobes (eg, sequences that are upstream or downstream of the coding sequence).

For example, this nucleic acid fragment can be used as a probe to identify other proteins of the MTI family. When used as a template, this nucleic acid fragment can be used as a template using the sequence SEQ ID NO. Designed by Molecular Cloning: Laboratory Manual++ (ed. Sula, Cold Spring Harbor Laboratory Publishing Department, 1985). It may also be a degenerate sequence. Therefore, oligonucleotide or nucleic acid flag The statement may contain all or part of the DNA sequence of SEQ ID NO: 1, and It may be a sequence biosynthesized based on the DNA sequence of SEQ ID NO: 1. This oligo The nucleotides are preferably suitably labeled using conventional labeling techniques.

The oligonucleotide also hybridizes with the mRNA transcript encoding the MTI protein. This complementary sequence, including sequences that An antisense sequence, called an antisense sequence, consists of all or part of the sequence of SEQ ID NO. The DNA sequence of SEQ ID NO: 1 may be used as a template). It may also be a designed biosynthetic sequence.

As yet another feature, the present invention provides gene sequences encoding proteins of the MT2 protein family. An oligonucleotide is provided that hybridizes to the column. This fragment has , identified antisense Nishikitari and MT2 line proteins, and/or Dimensions 11 - Sequence forces useful as probes for identifying coding sequences (including be caught.

The hybridizing nucleic acid contains all or part of the sequence of SEQ ID NO: 3. Also, a biosynthetic sequence designed using the DNA sequence of SEQ ID NO: 3 as a template can be used. columns, preferably suitably labeled using conventional labeling techniques.

The gene sequences identified herein are indicative of tissue malignancy or other cellular dysfunction. encodes a protein that is identified as a marker protein that induces Therefore, with another characteristic The present invention provides for the use of the proteins disclosed herein in combination with appropriate adjuvants. and to obtain antibodies useful for detecting cancer marker proteins in samples. provide products. In another aspect, the invention provides mRNAs encoding these proteins. Provides genetic templates for designing sequences that specifically hybridize with transcripts provide In yet another aspect, the invention provides epitopes of MTI or MT2 and specific In order to design binding proteins (including antibodies) that react with Provides isolated DNA sequences for use in expressing the fragments. The present invention is In addition, the gene sequences disclosed herein and the markers encoded by them A method for testing tissue conditions using proteins is provided. Finally, the present invention These marker proteins or their A method to treat malignant diseases using individual gene sequences encoding as target molecules. provide law.

Ward Saratate Suzukasa Ward Gai Figure IA-ID is a schematic diagram showing the amino acid sequence of MTI of SEQ ID NO: 1 as follows. be: Figure IA Nibroline residue position; Figure IB: Region defined as α-helix within the sequence; Figure 1C: Position of the Instein residue. Place; Figure LD: Cleavage site of NTCB.

Figures 2A-2B are schematic diagrams showing the amino acid sequence of MT2 of SEQ ID NO: 3 as follows. The diagram is: Figure 2A: Position of nibroline residue; Figure 28: Regions defined as α-helices within the sequence.

Figure 3 shows body fluid soluble MT measured in various normal and malignant tissue sample supernatants. 2 and MT2-related protein amounts are shown.

Figure 4 shows the body fluid flux measured in serum isolated from cancer patients and normal blood donors. The amount of soluble MT2 and MT2-related proteins is shown.

Lord 1” 1 mile Examining the properties of INM protein useful as a malignant cell marker in biological anasthesia In an attempt, the gene sequences encoding two types of INM proteins (herein referred to as MTI and MT2) was identified and its properties were investigated. The DNA sequences encoding these proteins are Isolated INM3 [expressed using monoclonal antibodies against MTI and MT2] cloned by searching the library.

This fluorescent white is essentially the method of Penman & Fey.

Nos. 4,882,268 and 4,885,236, incorporated herein by reference. The malignant cells were isolated according to the method (included in the specification). Cloned DN A was subsequently sequenced and its reading frame identified and analyzed. Gene encoding MT2 Child sequences have been disclosed by others (Yang et al. (1992) J, Ce1l Biol, plates 1303-1317 and Compton et al., (1992) J. Ce1l Riot, plate 1395-1408), by them "NuMA" being called. By comparing MTI and MT2 with other sequences in the art, The sequences encoding these proteins share little homology with previously reported sequences. It was shown that they form an array.

MTI was also expressed as a cleavable fusion protein in E. coli and isolated from mammalian cells. compared with that of the protein. Anti-MTI antibodies against naturally occurring MTI proteins also It showed cross-reactivity with the protein produced by the transgenic plant. Naturally derived proteins and recombinant production Both proteins had the same apparent molecular weight (90 kD) when examined by 5DS-PAGE. ), and both proteins have pI values (5, 4) similar to those of 2-nitro-3-thiosyl Cleavage with anobenzoic acid (NTCB, see below) shows the same cleavage pattern.

Data from MTI immunolocalization experiments indicate that MTI protein is localized in non-mitotic cells. As localized patchy follicles within the INM, they are unevenly distributed in the cough mass of the INM. Ru. In particular, this follicle exists between the chromatin of the nucleus and acts as an internal nuclear matrix protein. As expected, it is distributed as stable bonds that remain even after chromatin extraction.

Furthermore, MTlil cells are excluded from the kernels and laminae. Not only that, but also During mitosis, the distribution of MTI changes and is arranged in a star-shaped pattern on the spindle of dividing cells. Ru. Although this protein does not occupy a fixed site in cooperation with the chromoseme, it suggest that MTI may play a structural role during mitosis. immunological station The data from the naturalization experiments are based on any known DNA-binding motifs (e.g. leucine dinoplasts). -) MTI amino acid sequence analysis data that cannot find any structural homology with I will do it.

MT2 (NuMA) protein is not expressed recombinantly, but on the other hand, this protein Predicted molecule ff1238kDa (calculated from the predicted amino acid sequence (see SEQ ID NO: 3) ) is compatible with that of naturally derived proteins.

Immunolocalization experiments of MT2 (NuMA) revealed that this protein is also non-mitotic. It forms spots, follicles that are present throughout the nucleoplasm of the cell, and is also excluded from the kernel. It shows. During mitosis, this protein moves to the poles of the mitotic spindle in dividing cells. It is. The secondary sequence is a coiled coil for folded proteins. d-con) motif (Compton et al., (199 2) J, Ce1l.

Biol, 1395-1408; Yang et al. (1992) J, Ce 11. Biol, 1li1303-1317).

■、Katahiro Tsuji The nucleic acids disclosed herein can be used to identify malignancy or other cellular abnormalities in cells. It encodes a fluorescent protein that is originally distinguished as a useful marker protein. Specifically, this Significant increases in these protein levels occur in malignant cells and in extracellular fluids (e.g. serum) of cancer patients. (PCT Publication No. 093109437 and see below) For example, the presence of these proteins or their transcripts in cells or samples containing nuclear remnants. The presence and/or abundance of malignant cells or other abnormalities (e.g. It can be used to determine whether a cell contains cells with a chromosomal abnormality (e.g., chromosomal abnormality). can. This sample can be an exfoliated cell sample or a body fluid sample, e.g. blood, blood. serum, plasma, urine, semen, vaginal secretions, spinal fluid, saliva, ascites, peritoneal fluid, sputum, tissue swabbing , a sample containing biological exudate (eg, breast exudate).

Furthermore, since INM proteins are released in soluble form from dead cells, marker molecules are not of interest. It can be used to examine the viability of certain tissues. For example, the marker protein , to monitor the release of these marker molecules into body fluids over time. The effectiveness of a medical condition or a therapeutic measure or method can be determined by

Particularly useful body fluids include blood, serum, plasma, urine, semen, vaginal secretions, 1FItl fluid, saliva. fluid, ascites, peritoneal fluid, sputum, tissue swabs, and biological exudates (e.g. breast exudate) It is. Methods for implementing these answers are described in U.S. Pat. No. 4,882,268 and No. 4,885,236 and U.S. Patent Application No. 214,022 (June 30, 1988). Hinode 1jJ and No. 785804 (filed on October 31, 1991) (these All references are herein incorporated by reference.

All of these annosées have the following general operating process characteristics: 1) “Marker proteins in authentic samples or reference samples or detecting the presence and/or abundance of that transcript; 2) presence and/or abundance of the marker protein or its transcript in the sample in question; Detect wealth; 3) Amount of marker protein or its transcript in the sample in question and reference sample Compare with the amount in the pull.

When using this assay to monitor tissue viability, the sample in question The step of detecting the presence or abundance of marker proteins or their transcripts in repeat, then compare these values. Changes in detected concentration indicate tissue condition reflects changes in Use this assay to examine the effectiveness of treatment If the drug or therapy is administered, the monitoring step should be performed after the therapeutic agent or therapy (e.g. after chemotherapy or radiation therapy).

Meaning that certain INM marker molecules are present in target cell types and not in others , the selectable marker protein or transcript need not be totally unique; However, the marker molecules in the sample are pre-selected for which the marker is designed. It is required to have a signal to noise ratio high enough to distinguish between cell types. . For example, the MTI and MT2 proteins, even if the proteins or their close analogs Even though they are commonly present in non-malignant cell types, their expression in malignant cells Because of the increased levels of be.

A brief discussion of protein and nucleic acid analysis considerations is provided below. identification Further details of the annoise conditions can be found in the annoise references cited above (these are incorporated herein by reference). published protocols well known in the art (these are readily available). (available at ).

A, ■ Wataru Etachiji Characterization of MTI and MT2 proteins at the molecular level as disclosed herein This has made it possible to characterize the protein structurally and biochemically.

Therefore, following the disclosure of these gene sequences and the amino acid sequences they encode, and preferred binding epitopes that can be used to enhance anisotropic conditions. can be identified. For example, binding proteins are produced by specific cell types. or affinity for marker proteins as a specific malignant function. can be designed to enhance Similarly, binding proteins are released from dead cells. They can be designed to bind preferentially to protein fragments.

Additionally, structural and/or sequence variations between proteins produced in normal and abnormal tissues can be clarified and utilized. The gene sequence is determined by standard methods known in the art. Transgenic DNA can be modified as desired using DNA manipulation, e.g., truncated or mutated. can be manipulated to obtain proteins with desired characteristics useful for antibody production. .

As will be understood by those skilled in the art, it is difficult to specifically identify and define a marker protein of interest. Any method of weighing can be used. The current preference for detecting the protein of interest in a sample is A preferred method uses a binding protein that can specifically react with the marker protein. It is something to do. mm antibodies, and especially binding portions thereof, can be advantageously used. . Antibodies may be monoclonal or polyclonal in nature, and may be produced biosynthetically. The amount of marker fluorescent complex, e.g., the amount of marker protein bound to the binding protein, may be determined by , subsequently determined using standard protein detection methods well known in the art. Ru.

A.1. I'm sorry for the inconvenience. A variety of different immunomodulatory forms currently exist, none of which are IN It can be used to detect and quantify MW white and its protein fragments. Ru. For example, for exfoliated cell samples, collect the cells and surrounding fluid and [ NMW white is produced by the method of Penman and Huey (U.S. Pat. Nos. 4,882,268 and 488). 5236). These proteins are then preferably converted into secondary The original gel was separated by gel electrophoresis and the presence of marker proteins was detected by Western profiling. Detected by the method of detection.

The marker protein and/or protein fragment to be detected is mainly present in the solution. Existing serum and other fluids are currently the most sensitive immune assays. The style is the Sanderutsch method. This method, which typically has an accuracy of ±5%, As disclosed in PCT Publication No. 093109437, Two antibodies are used that are capable of binding the analyte: for example, one is attached to a solid support. It can be easily detected whether it is fixed on the carrier and the other is free in the /8 liquid. labeled with chemical compounds.

Examples of chemical labels that can be used for the second antibody include radioisotopes, fluorescent When brought into contact with photocompounds and enzymes, or reactants or enzyme substrates, a colored product is produced. compounds or other molecules that produce electrochemically active products. marker When a sample containing a protein or its protein fragment is introduced into this system, the marker The car protein binds to both immobilized and labeled antibodies.

The result is a 'sandwich' immune complex on the surface of the support.

This complex protein washes away unbound sample components and excess labeled antibody and supports the It is detected by measuring the amount of labeled antibody that has formed a complex with body surface fluorescence. Ru. Sandwich immunoassays can be performed using labels with excellent detection limits. , very specific and very sensitive. Immunological answer design, theory and A detailed review of the protocols and protocols can be found in “Practical [Immunology]”. mmunology)’d, R, Butt klA, Marcel Dek ker, New York, 1984). Can be done.

In general, considerations for the design of immunoassays include antibodies (e.g., monoclonal antibodies or involves the preparation of polyclonal antibodies), which specifically bind antigen-antibody for those antigens that can reliably distinguish complex proboscis from non-specific interactions. has sufficiently high binding specificity. As used herein, an "antibody" , other binding proteins with appropriate binding affinity and specificity for the marker protein. It is considered to include. The higher the binding specificity of the antibody, the lower the antigen concentration that can be detected. becomes lower. Currently, the preferred binding specificity is that the binding fluorescence is greater than about 10'M-', preferably greater than about 10'M-' binding affinity.

Antibody binding domains can also be produced biosynthetically, by modifying the amino acid sequence of the binding domain. Can be manipulated to enhance binding affinity with preferred epitopes. MTI and Identification of the gene sequences of M. and M.T.2 provides advantages for the design and construction of preferred binding proteins. For example, DNA encoding a preferred epitope can be used for Mi to select antibodies that selectively bind to this epitope. It can be used for Subsequently, the specific cough matrix protein or protein Sample the selected antibody under conditions sufficient to cause specific binding of the antibody to the fragment. and the amount of complex formed is then detected. About specific antibodies The methodology is well known and described in the literature.

A more detailed description of its preparation can be found, for example, in "Immunological Practice" [(Pract ical Immunology) L R, Butt kXA, Marcel Dekker, New York, 1984).

The choice of label also depends on the desired detection limit. Enzyme analysis (ELISA) is standard. Typically, a colored product formed by the interaction of an enzyme-labeled complex with an enzyme substrate Let objects be detected. Other labels include radioactive or fluorescent labels. Until today The most sensitive labels known are chemiluminescent labels; The interaction results in the generation of light; useful labels include chemiluminescent molecules (e.g. acridium). ester) or chemiluminescent enzyme (in which case the reactant is the enzyme substrate). Ru. For example, when acridium ester reacts with an alkaline peroxide solution, a strong A flash of light is emitted and the detection limit is 100 to 10,000 times higher than that obtained with other labels. increases the detection limit. Furthermore, this reaction is rapid. chemiluminescence and immunology A detailed description of the assembly can be found in Weeks et al., (1 983) Methods in Enzymology ) Yu 366-387], other considerations for liquid annosé include microtiter ) Includes the use of well or column immunoassays. Column answer is quick ilr reacts to! a, it is particularly advantageous if, for example, chemiluminescent labels are used. mark The identification complex can be eluted post-column detector, which also contains reactants or enzymes. The elementary substrate is included and the subsequently formed product can be immediately detected.

A.2. disease production The proteins disclosed herein are based on standard immunological techniques that are well known and described in the art. can be used to produce antibodies using standard methods.1 For example, Practical Epidemiology J (edited by W. R. Butt, Marcel Dekker, 2 S-York, 1984) 1. Briefly, for example, if a recombinant is produced in a host cell, Isolated INM protein produced by expressing DNA is used to raise antibodies in a heterologous host. Used for production. Preferred antibodies specifically bind to epitopes on the protein. antibodies (preferably with a binding affinity greater than 10'M-' for the epitope) , most preferably with a binding affinity of greater than 10'M-'). for example , H) If antibodies against INMi white (e.g. MTI or ~IT2) are desired. suitable antibody-producing hosts include mice, goats, rabbits, guinea pigs, or Other mammals used for body production. The protein enhances antibody production in the host into the host, e.g. by intraperitoneal administration. Can be injected. Any adjuvant appropriate to stimulate the host immune response. Bunts may also be used for convenience. Currently preferred adjuvant Complete Freund's adjuvant (an emulsion containing sterilized and dried microbial cells) [For example, Calbiogam (San Diego) or Gibco (Grand Island) , New York) 1. If multiple injections of antigen are desired, subsequent injections may be unnecessary. Contains antigen mixed with complete adjuvant (eg, cell-free emulsion).

Polyclonal antibodies are produced by extracting serum containing antibodies against the protein in question from an antibody-producing host. It can be separated by taking it out.

Monoclonal antibodies are produced by isolating host cells producing the desired antibody and are used in the field of immunology. These cells were fused with myeloma cells using a method known in Hyprinted cells (hyprinted cells) that specifically react with INM protein and have the desired binding affinity It can be produced by screening for hybridomas).

Provided below are currently preferred monoclonal antibody production protocols. This is an example. Other protocols are also contemplated. Therefore, the cancer marker of the present invention Specific antibody production methods for car protein compositions are contemplated as a feature of the present invention. Not really. Also described below detects and detects marker pallor in samples. / or useful for quantifying sandwich immunoassays and dontobronots This is an example of an answer. Marker proteins (especially MTI, MT2 and their analogues) other means of detecting protein fragments and naturally occurring variants) is also intended. These other methods are well known and described in the art.

However, protocol: Ba1b/cXJ mouse (Jackson Laboratory, Barba Purified IN purified from a human cervical cancer cell line (CaSki) M protein (eg, MTI) is injected intraperitoneally every two weeks for 16 weeks. to the mouse A single booster injection is given 4 days before sacrifice, and the pancreas is removed. at the first injection Second time using Freund's complete adjuvant (Gibco, Grand Island) Incomplete Freund's adjuvant was used for the first injection, and subsequent injections were performed with saline. give Pancreatic cells (or lymph node cells) were then collected using the literature method [Kohler & Milstein (1975) Nature 495 (see this document) (herein incorporated by reference) 1, polyethylene glycol (PEG: base) fused with a mouse myeloma cell line (Linger Mannheim, Germany).

Then, hybridomas producing antibodies that react with nuclear matrix proteins are cloned. rowing and proliferate as ascites. using standard methods known in the field of immunology. The cell line from which the immunogen was derived was tested for nuclear reactivity and tissue immunochemistry. Screen for bleedomas. Screening protocols, ascites production and An immunoassay is also described in PCT Publication No. 193109437.

Betsu M-Toni-ni A. Sandwich immunoassay (ELISA) for antigen binding and cross-reaction analysis , and standard for creating dose-response curves for monitoring tests. Immunology tests can be performed. The data are based on standard preparations of NM antigen. It is used as a reference standard when analyzing body fluids. In these examples the ELI Both SA and radioimmunoassay were performed.

1. Immunology analysis (well analysis) Microtiter plate (Imron TI, Guinatenku, Nyantiri, Ba Genia) with purified antibody at 5 to 15 Mg/ml (in PBS, pH 7.4). Cover for 1 hour or overnight, then wash 3 times with 300 μl PBS. Then press Rates were blocked with 10% normal goat serum in PBS for 1 hour at room temperature and then incubated for an additional 30 Wash 3 times with 0 μl of PBS. An example of the protocol is shown below.

Here, pipette a 100 μm sample per well and keep it at room temperature for 1 hour. Warm and analyze. Wells were washed three times with 300 μl of PBS. 1.25 to 1 Add 100 μl of biotinylated antibody at 0 Mg/ml to each well and incubate for 1 hour at room temperature. The cells were kept warm for a while and washed three times with 300 μl of PBS. 1:1000 times diluted Strep Toavidin horseradish peroxidase complex (Pinding Sight, Inc., Birmingham, UK) was added to each well, incubated for 1 hour, then Washed with PBS. Then 100 μl of peroxidase substrate (citrate, Add phosphate, OPD (HzOz) to each well and incubate for 20 minutes.

The reaction is stopped by adding 50 μM of 1M sulfuric acid to the wells. Optical density plate Read at 490 nm with a scanner.

To compare with unknown samples, prepare reference concentrations of antigen and perform standard dilutions. Determine antigen concentration by preparing a line.

2. TRMA・Ace (a) Iodination of streptavidin 10 Mg streptavidin in 2 μl 0.05 M phosphate solution (pH 7.4) (Sigma, Cincinnati) in a microcentrifuge tube with 10 μl of 0.25 M phosphate solution (p H7, 4), in addition to 10 μl of 1 mC1 “’ , Wilmington, Plowea). 100 in 50 ml of distilled water immediately Add 10 μl of mg chloramine-T trihydrate (Sigma), mix and react for 25 seconds. let followed by 40 mg cysteamine (2-mercaptoethylamine, Sigma ) and 5 mg in 50 ml of 0.05 M phosphate solution (pH 7.4) Mix KI for 20 seconds to stop the reaction. 1% B in PBS (pH 7,4) Add 0.5 ml of SA and transfer the material to a 10 ml volume previously equilibrated with BSAPBS buffer. Fractionation was performed on a Sephadex G-100 column (Pharmacia, Sweden). 0 ．． Collect 5ml x 30 fractions and dilute lμl of each fraction to 1ml with BSA/PBS. Dilute.

Count 100 μl of the diluted fraction using an LKB gamma counter for +isI. ratio The activity was calculated and was usually between 85 and 100 μC4/μg. Inside the protein peak The central fraction is then used in a sandwich immunoassay.

(b) Sand Inch Radioimmunoammonia-Microtiter Separate Well (Immuro II Remover Well Strip, Dynatic, Chantilly, Virginia ) to EL[SA Anonymous, and then) long.

Samples (standards or serum) are routinely measured as follows. 1 in the well Incubate 00 μl at room temperature for 1 hour, then add 300 μl of PBS using a plate washer. Washed three times, followed by biotinylated antibody (2-10 Mg/m in 10% goat serum + ) for 1 hour at room temperature and wash again. The bound biotinylated antibody was Detect with treptavidin. 200,000 to 300,000cp in 100μl Add m (77% measurement efficiency) to each well, incubate at room temperature for 1 hour, and wash again. . The bound fraction is detected by measuring radioactivity using an LKB gamma counter. 4 degrees Counts obtained for reference preparations can be compared.

B, NM dot plot detection The reactivity of the antibody with the N~1 protein can be determined using standard methods and equipment (e.g., Sch. It can be investigated using a dot plot detection assay using can. The nitrocellulose membrane was soaked in Tris-buffered saline (TBS, 50mM TRl). 5. Soak in 150ffiM NaCl, pH 7,6) and inject seeds into a series of wells. Treat with NM#A product at different protein concentrations and incubate for 1 hour at room temperature (e.g., 10M g/ml, 1 μg/ml and 100 n g/ml of T-47 DNM supernatant), then the blocked wells were washed twice with 200 μM TBS, Then block with 100 μM 10% normal goat serum in TBS for 1 hour at room temperature.

The blocked wells were then washed again twice with 200 μM TBS and treated with nuclear activity antibody. Add 100 μl of test culture supernatant containing Ru. The wells were then washed twice with 200 μm TBS and treated with alkaline phosphatase. 100μ serial dilution of conjugated goat anti-mouse IgG] (Bio-Rad, 'J, Chi Mondo, California) (e.g. 1:1000.1:5000, or 1 : 10,000) was added to each well and kept warm for an additional hour. continue The wells were washed twice with 200 μm TBS and further treated with levamisole (Hector, Inc., Enzyme substrate (Corpus Christi, Texas) in Tris buffer containing BCIP/NBT, Kirkgaard & Perry, Gaithers burg, MD, e.g. 100μ+). Generally, keep warm for 15 minutes It is enough. The reaction can be stopped by washing with distilled water and the purified product can be spewed out. .

B. By detecting the amount of transcripts encoding these cancer marker proteins, tissue It is also possible to determine the state of .

To date, the preferred means of detecting mRNA has been the use of labeled oligonucleotides. (e.g., a nucleic acid fragment that can specifically hybridize with the transcript of interest). This is the Northern plot analysis method used. Preferred oligonucleotides so far The octide sequence encodes a sequence that is complementary to at least a portion of the marker sequence transcript. This is an array. These complementary sequences are known in the art as "antisense" sequences. It is known in the art. This oligonucleotide is an oligoribonucleotide or It would be an oligodeoxyribonucleotide. Furthermore, oligonucleotides are naturally Oligomeric and biologically important nucleotides (i.e. A (adenine), dA (deoxyadenine), G (guanine), dG (deoxyguanine), C ( cytosine), dC (deoxyadenine), T (thymine), U (uracil)), or, for example, to methylate the phosphate oxygen in the phosphodiester bond between nucleotides. composed of modified oligonucleotide species in which a sulfur group or a sulfur atom is substituted (e.g. If the following 1. (see Chapter C), in addition to the nucleotide itself and/or its The repose portion may also be modified.

This sequence is a known chemical oligonucleotide well-described in the art. Can be chemically synthesized using any of the following synthetic methods: For example, oligonucleotide can be advantageously prepared using any commercially available automated nucleic acid synthesizer. . Alternatively, oligonucleotides can be prepared using standard recombinant DNA techniques, e.g. can be produced by inducing transcription of the double strand. For example, marker protein The DNA sequence encoding white can be reversed in a recombinant DNA system. example For example, insert in the opposite direction downstream of the appropriate promoter so that the non-coding strand is transcribed. can.

Useful hybridization oligonucleotide sequences include MTI or MT2-transfer Includes any sequence that can specifically hybridize with the transcript. Therefore As will be appreciated by those skilled in the art, useful sequences included are SEQ ID NO: 1 (MTI ) or SEQ ID NO: 3 (MT2) (MTI and MT2 are protein coding regions and In addition to the DNA sequences provided by or downstream transcribed sequences (e.g., those contained in the 5° and 3° untranslated regions, or or sequences extending into said region) and complementary sequences. Typical antise The sequence sequences are set forth in SEQ ID NOs: 5 and 6. Sequence number 5 is MTII white The first 100 nucleotides of the code sequence as well as the 53 nucleotides upstream of the start codon Represents a sequence complementary to a nucleotide sequence. Nucleo complementary to start codon Tido is located at positions 298-300 of SEQ ID NO:5. Similarly, SEQ ID NO: 6 is MT2 The first lOO nucleotides of the protein coding sequence (compare SEQ ID NOs: 3 and 6) ) as well as a sequence complementary to the 48 nucleotides upstream of the start codon , the nucleotides complementary to the start codon are located at positions 298-300 of SEQ ID NO:6.

Useful oligomers are based on part or all of the sequences SEQ ID NOs: 5 and 6. It can be made by However, it will be understood by those skilled in the art that However, other useful sequences that hybridize with other regions of the transcript also include SEQ ID NO: 1 and and 3, and/or another untranslated sequence (e.g. Compton et al. Disclosed about M T2 (N u M A ) of Yang et al.) and Yang et al. can be easily created based on sequences existing in

Oligonucleotides of any length can be used to hybridize with mRNA transcripts. Although sequences shorter than 8-15 nucleotides can be used to target Specificity may be low when hybridizing with mRNA. Therefore, the standard Typically oligonucleotides within 8-100 nucleotides, preferably 15- Within the range of 50 nucleotides, standard RNA hybridization It is thought that this would be the most useful.

Oligonucleotides selected to hybridize with INM transcripts whether synthesized chemically or with recombinant DNA) is then labeled. isolated and purified using standard techniques, and further purified using standard labeling protocols. is labeled (eg with 35S or 32P).

Samples containing the marker transcripts of interest were subsequently run through an electrophoretic gel and dispersed. Transfer the nucleic acids to a nitrocellulose filter and add the 1m oligonucleotide to an appropriate halide. Contact with the filter under industrial conditions. hybridization The run conditions are “＼ Rowning: Manual J [(Molecular Cl oning: A Laboratory Manual), Mania-Nice et al. For example, 50% formamide, 5X SSPE, 2X Den Halton Yong 7F! L, 0.1% SDS, 42°C. known in the art Other useful methods include solution hybridization and dot and slot hybridization. Includes RNA hybridization. Next, marker transfers present in the sample are The amount of transcripts is flagged using standard methods known in the art. Quantitate by measuring the radioactivity of the ment.

Following a similar protocol, we also coded for the types of MTI and MT2 protein systems. oligonucleotides to identify other sequences (e.g., as described in the Examples below). Nucleotides can be used. This methodology is based on the protein components disclosed herein. To identify gene sequences related to a protein coding sequence, e.g. A non-coding sequence that exists in or downstream of these genes and has a functional role in the expression of these genes. It can also be used to identify potential players. new ma degenerate sequences and/or preferred codon bias when identifying one type of carrier. using the sequence of SEQ ID NO: 1 or 3 as a template) and further degenerate. be prepared using general guidelines disclosed in the art dealing with Can be done. 1 For example, “Molecular Cloning: Laboratory Manual Sula) See 〇　.

The proteins disclosed herein are associated with the mitotic spindle apparatus and increase in abundance in malignant cells. Add. Therefore, without being bound by any particular theory, these proteins play a role in cell division. It is assumed that the structure plays an important role in the therefore, these proteins and their transcripts may be useful for cancer chemotherapy. This would be a preferable candidate as a target molecule.

C, 1E earth + 7 prisoner blood Particularly useful cancer therapeutics envisioned are those that are complementary to part or all of the marker transcript. an oligonucleotide that specifically hybridizes to the transcript and when hybridizing with the mRNA transcript, the limRNA Translation can be suppressed. Antisense oligonucleotides are normal and It has been widely used to suppress gene expression in abnormal cells [e.g. For a comprehensive review of the relationship between theory and theory, see Stein et al. ancer Res, 48:2659-2668) and existing protocols. See the rules 1. Therefore, antisense antibodies against MTI and MT2 Using cleotide as part of chemotherapy, alone or in combination with other treatments can be done.

1 above. As mentioned in Section 8, oligoribonucleotides and oligodeoxynucleotides Both of the nucleotide sequences hybridize with the mRNA transcript and are described herein. It can be used to suppress the translation of mRNA1 of the marker protein disclosed in Probably.

However, oligoribonucleotides are generally more useful than deoxyribonucleotides. Highly susceptible to enzymatic attack by ribonucleases. Therefore, oligodeoxy Liponucleotides can be used for in vivo therapy to suppress mRNA translation in individual patients. preferable.

Also, the above 1. As mentioned in Section B, the therapeutically useful antisense of the present invention Oligonucleotides are known chemical oligonucleotides that are well described in the art. It can be synthesized by any of the cleotide synthesis methods. Alternatively, the natural mRNA sequence Sequences complementary to a portion or all of the It can be made. For example, the DNA encoding this protein coding sequence This non-coding strand is placed in the reverse direction downstream of a promoter that can express the sequence. It can be inserted to be transcribed.

The complete nucleotide sequence of this protein-coding sequence is Similar translation sequences are known for both MTI and MT2 (e.g. SEQ ID NO: 1 and 2 and Compton et al.), and also using the present disclosure. It is possible to determine whether any part of the mRNA transcripts of these proteins Antisense oligonucleotides that can also hybridize are readily available to those skilled in the art. can be prepared using conventional oligonucleotide synthesis methods known in the art.

complementary to any part of the MTI and MT2 mRNA transcripts and hybrid Oligonucleotides that can be redized are transferred as disclosed herein. It is generally effective in suppressing the translation of photographs. For example, US Pat. No. 5,098,890 (19 Effective March 24, 1992, this document is incorporated herein by reference. The origin complementary to the mRNA at or near its translation initiation codon site is The use of gonucleotides is advantageous for translational suppression. Furthermore, from the translation start site Sequences that are too far apart in the 3° direction limit potential “read-through” of the liposome. antisense/sense duplexes (dublens) to enable translation of For phenomenon 1, which requires liposomes to unravel The hybridization effect with the photo is low.

A representative antisense sequence for MTIδ and MT2 transcripts is SEQ ID NO: 5 ( MTI) and SEQ ID NO: 6 (MT2). This antisense sequence is In addition to the sequence encoding the N-terminus of either the MTI or MT2 marker protein , is also complementary to the 5' untranslated sequence immediately upstream of the start codon (details of these sequences can be found here). For detailed explanation, see 1. above. (See Section B).

As will be understood by those skilled in the art, other than MTI and/or MT2 transcripts For example, antisense oligonucleotides complementary to the region of SEQ ID NO: 1 and It can be easily produced using the sequences shown in 3 and 3 as a template.

1 above. As mentioned in Section B, oligonucleotides of any length can be Can be used to hybridize with transcripts. However, very short Binding of sequences (eg less than 8-15 nucleotides) may be less specific. So Above, for in vivo use, such short sequences are particularly sensitive to enzymatic degradation. Furthermore, if the oligonucleotide is given directly to the cells, it may be very long The sequence will have less inhibitory effect due to reduced uptake by target cells. did Therefore, when giving oligonucleotides directly to target cells, 8-50 nucleotides oligonucleotides, preferably having a length in the range of 15-30 nucleotides. It is assumed that Otido is the most advantageous.

Another means of delivering antisense sequences to target cells is as part of gene therapy techniques. , for example, a protein capable of expressing an antisense sequence preferably constitutively in target cells. It is present as a DNA sequence (preferably part of a vector) with a promoter.

Recently, Oeller et al. (1992), Company 43 7-539; this document is incorporated herein by reference) is the full-length AC N that can be expressed constitutively and encodes an antisense sequence for C-ntase transcription. reported in vivo inhibition of ACC synthase using the A sequence. Therefore, an If the sequence is provided indirectly to the target cell, e.g. When provided as part of an expressible gene sequence, substantially all of the protein-coding sequence It is advantageous to use longer oligonucleotide sequences that contain complementary sequences for all Probably.

Finally, above 1. B! As also mentioned in ff, the postulated therapeutically useful Oligonucleotides are natural oligonucleotides composed of naturally occurring nucleotides. sesame, but also improves e.g. stability and lipid solubility, thereby improving cellular uptake. Oligomers containing modified nucleotides with enhanced loading are also included. In fact, enhanced solubility and/or enhanced nuclease resistance can be The phosphate oxygen in the phosphodiester bond within leotide is replaced by a methyl group or sulfur It is known that this can be achieved by substituting atoms. Phosphorothioe (“S-oligonucleotide” in which the phosphate oxygen is replaced with a sulfur atom) °“) are stable against nuclease cleavage, lipid soluble, and especially oligonucleotides. Preferred S-oligonucleotides for direct administration of cleotides are those mentioned above. ．． It can be chemically synthesized using known automated synthesis equipment as described in Section B.

Oligonucleotide sequences that effect mRNA translational repression are described herein and Additionally, standard in vitro methods using well-characterized standard methods in the art. Easily evaluated in Roanose. An example implementation of the protocol is described below, but other protocols Rotcols are also expected to be used to advantage.

Antisense candidate sequences can be generated as provided herein using standard chemical techniques. For example, M having the sequence shown at positions 285-315 of SEQ ID NO: 5. TI antisense sequences were generated using an Applied Biosystems automatic DNA synthesizer. The oligonucleotides can be purified according to the manufacturer's instructions.

The oligonucleotides are then cultured in a suitable malignant cell line (e.g. ME~180). under standard culture conditions and allowed to be taken up by the expanded cells.

Preferably, a range of dosages is used to achieve similar hybridization specificity. Determine the effective concentration for inhibition, e.g. o-too μg oligonucleotide. Dosage ranges of 0.0/ml can be investigated. Additionally, oligonucleotides are only DNA can be given to cells in a single transfection, but It can also be given as part of a continuous transfection.

The antisense effect increases cell proliferation over time after transfection. Changes in growth can be detected using standard cell counting methods and/or decreased expression of marker proteins. For example, I above.

It can be analyzed by immunofluorescence as described in A. Separately, cells The ability to incorporate and utilize thymidine is another I! in the analysis of cell division. car hand This step can also be carried out using, for example, 3H thymidine. effective a antisense inhibition sufficiently suppresses cell division and reduces thymidine incorporation. , inhibit cell proliferation, and/or reduce detectable levels of marker proteins. It should be possible.

Useful concentration ranges depend on the nature and extent of the neoplasm and the particular oligonucleotide used. , according to the relative susceptibility of the neoplasm to oligonucleotides and other factors. It is assumed that the amount will fluctuate.

Useful ranges and oligonucleotides for a given cell type can be determined by standard dosing. Range experiments can be performed as described herein to determine. dosage range actual Experiments can also be performed to determine the level of toxicity against normal and malignant cells.

Advantageously, a dose of about 1 to 100 μg/ml per 10S cell is used.

For in vivo use, antisense oligonucleotides may be carried in a pharmaceutical carrier (e.g., a suitable liquid excipients) and optionally auxiliary additives. liquid supplement The formulation is conventional and commercially available. Examples of these are distilled water, physiological saline, Examples include glucose aqueous solution. Antisense sequences are preferred for in vivo cancer therapy. Preferably, it is given directly to the malignant cells, for example by injection at the neoplastic site. Another thing In addition, the antisense sequence is accompanied by a means of directing the sequence to the target malignant cell. Optionally, the oligonucleotide can be administered systemically.

In addition to being administered with conventional carriers, antisense oligonucleotides can be administered in a variety of ways. Can be administered by specialized oligonucleotide delivery techniques. For example, Ligonucleotides can be encapsulated by ribosomes as described in the literature. [Maniatis et al.; Mannino et al. (1988) Bio Technology, 682; Feigner et al. (1989) BeLh esda Res, Lab, Focus, n:211. Outside the reconstructed virus Wi (envelope) is also used to deliver RNA and DNA to cells [For example, Arad et al. (1986) Biocell, Bi ophy, Acta, Takumi 88-94].

For therapeutic use in vivo, antisense oligonucleotides have therapeutic potential. given in an effective amount, e.g., an amount sufficient to inhibit target protein expression in malignant cells. Ru. The actual dosage administered will depend on whether the treatment is prophylactic or curative in nature, the age of the patient, Body weight, health and gender, route of administration, size and nature of malignancy, as well as other factors. can be considered. The daily dosage ranges from approximately 0.01 to IO0 per day. A range of 0 mg is possible. Higher or lower amounts of oligonucleotide may also be used. It can be administered as desired. It will be understood by those in the medical field, especially in the chemotherapy field. Therefore, the appropriate dosage range for in vivo administration is a routine test for clinical laboratories. It would be an investigation. As a preliminary guideline, as described above, in vitro It is possible to lower the concentration that is effective for inhibiting oxidation.

11, B] L blood and■ In another example, the cancer marker protein itself can be used as a target molecule. For example, binding agents designed to bind marker proteins essentially irreversibly. For example, if a white substance is specific to cells and is known to be absorbed by cells. It can be delivered to malignant cells by binding it with a corresponding ligand. Special Methods for targeting molecules to specific cells and cell types are well-described in the chemotherapy field. It is.

The binding protein is 1. can be obtained and tested as described in section A. for example , binding portions of antibodies can be advantageously used. In particular, those that use A binding protein that is identified with a high affinity, e.g., an affinity of greater than about 10"M-' It is white.

Alternatively, the DNA encoding the binding protein can be prepared using a genetic method well described in the art. Expressable genes to be expressed within cells after manipulations used in child treatment protocols as part of the target cell (e.g., U.S. Pat. No. 4,497,799). No. 6 and “Gene Transfer” (Vijay R, (see Baichwalm, (1986)). The INM protein complex is It is expected that this will be able to impede and suppress cell division.

As mentioned above for antisense nucleotides, for in vitro use For this purpose, a suitable conjugated fluorophore can be placed in a suitable pharmaceutical carrier (e.g. saline or medical solution). It can be combined with other carriers whose properties are well known in the field. The pharmaceutical composition is can be given directly to malignant cells, e.g. by direct injection, but also systemically, provided that the binding protein is accompanied by a means of targeting the protein to target cells. It can also be administered to Finally, appropriate dosage ranges and cytotoxicity levels should be determined by standard can be investigated using standard dose range experiments. A therapeutically effective concentration is 0. The actual dosage, as stated above, will be in the range of 1-1000 mg/day. Depending on other factors, such as the nature of the malignancy, the age, weight and health of the patient, will also change.

11.1 The following examples further demonstrate MTI and MT as markers for abnormal cell types. 2 and further describes the gene sequences encoding MTI and MT2 proteins. The method of isolation and characterization can be The best L for engineering and characterization! For example, INM in E. coli i white expression is disclosed herein. However, other prokaryotic and eukaryotic cells Current systems are also envisioned for recombinant expression of the proteins disclosed herein. included Other useful hosts include yeast, insect/baculovirus expression systems, and mammalian cells. (For example, Chinese hams with different types of myeloma cells and star ovary cell line).

Kasaeto As shown below, MT1 expression levels were found in breast, colon, bladder, ovary, prostate and significantly enhanced in a number of different malignant cell types, including malignant cell types of the uterine cervix. Ru. The results shown below are the results of a standard immunoassay (accuracy ±5%). Normal and malignant human tissue extracts (which include 8M as described in the PCT publication) Prepared with urea, 2% β-mercaptoethanol, and 2% Nonidenoto P-40 (detergent). manufactured) in this specification and PCT Publication No. W093103497. Performed as described.

The 302.47 antibody was derived from Ca5ki [cervical cancer cultured cell line (Averican)]. Type (ulture Co11ection), ATCC, Rockville, Maryland) origin N M1i! Made for products. MTI: 2-8 was created for the cloned MTI protein. Both antibodies use standard binding assays. The epitope on the protein encoded by SEQ ID NO. join with the group. As can be seen from the results presented below, MTI is associated with malignant bladder tissue. It increases markedly. Blond experiments also indicate that MTI levels are elevated in other malignant tissues. Which indicates that.

Table I First, the naturally occurring MTI protein was synthesized essentially from U.S. Pat. 8C in issue 5236! ! Human offspring according to the method of Venman and Huey It was isolated from cervical cancer. Human cervical cancer cell lines Ca5ki and ME180 [ Obtained from ATCC (Oy Kuville, MD) The cells were allowed to grow until they were fully grown and removed from the flask by trypung treatment. suspension cells Wash twice with phosphate buffered saline (PBS) and add cytoskeleton buffer (C3K: 100mM NaCl, 300mM Licorice, 10mM PIPES, 3mM MgC1z, 1mME Extract with GTA, 0.5% Triton-X100.1.2mM PMSF) at 4°C for 1 minute. followed by cold R5B (reticular cell suspension buffer) 72x detergent buffer (100mM aC1,3m　M　MG　CIz, 10mM) Lisu (pH 7,4), 1% H Extracted with Tween 40.0.5% deoxycholate, 1.2mM PMSF).

Separately, cells were extracted twice with RB S/2x detergent buffer. Two extraction protocols A very similar preparation was obtained by Coll. Gently digest the extracted cells for 30 minutes at room temperature. Solution [50mN a C! , 300mM Licorice, 00m% Triton-X100. 10mMP IP ES Cp H6,8), 3mMgC1g, 1m MEGTA, 1.2mM PMSF (100μg RNase and D (containing NNase). 2M ammonium sulfate to 0.25M final chromatin was extracted from the digested nuclei. extracted nuclear matrix - The intermediate filament (NM-[F) groove composition was then sedimented at 3700Xg for 15 minutes. I set it.

The resulting pellet was then added to a dispersion buffer (8M urea, 20mM MES (pH 6). , 6), 1mM EcTA, 1mM PMSF, 0.1mM MgC1z, 1% 2-methane sonicate the pellets and further volume of assembly buffer (0.15M KCl, 25mM imidazole (pH 7) , 1), 5mM MgCL, 2mM DTT, 0.125mM EGTA, 0.2mM The mixture was dialyzed overnight with three changes of PMSF). Dialysate followed by 10,100,000x hours After centrifugation, 8M protein was recovered from the supernatant. Alternatively, NMIF components can be directly added to E40. 0$1 solution (0,4M NaCl, 0,02M) Lis pH 7°5.0.1mM M gC1z, 0.5% 2-mercaptoethanol, 1.2mM PMSF) at 4°C. 30 minutes, R [von Krtes et al. (1991) Ce1 1, 64:123-135]. Next is Beckman 70. I After centrifuging for 90 minutes at 4000 rpm in a Ti rotor, the intermediate filament is enriched. Removed the beret. The remaining supernatant contains almost no cytokeratin contaminants and is MTI Rich in protein.

MTI-specific antibodies were generated using standard methods. In particular, Ba1b/cXJ mice (Japanese) Purified Caski NM protein (Cuman Laboratory, Bar Harbor, ME) for 2 weeks. Six intraperitoneal injections were given every 16 weeks. Mice were given 4 days before sacrifice and lung removal. One booster injection was given. Complete Freund's adjuvant was used for the first injection; The second 6 injections were given with incomplete Freund's adjuvant and subsequent injections were given with saline. provided. Lung cells were obtained from the SP210-Ag14 murine engineered cell line (ATCC, Kubill, MD) using standard fusion methods well known in the art. fused. Hybrid-7 producing antibodies that react with nuclear matrix proteins was cloned and grown as ascites. Antigen specificity was measured using an immunofluorescence spectrometer and I looked it up on Western Bronot Anse. 302.47 antibody as below. The expression library was screened and the MTI gene was isolated.

The cDNA clone of MTI was converted into a lambda ZAP expression library (Stratagene Co., Ltd., La Jolla, California).

Library screening was performed using the MTI-specific antibody 302.47 according to the manufacturer's instructions. It was carried out according to the instructions. Simply put, just one with a 2.45kb insert. Positive clones of Ec.

pBluescript hector opened at R1 and Xhol cloning sites (Strategene, La Jolla, CA). profit The sequence of the generated plasmid (pMTl) was directly examined and further subcloned. An MTI fusion protein was produced (see below).

c. DNA sequences were obtained using standard dideoxy methods described in the art. It was done. Double-stranded sequencing was performed by the manufacturer (Stratagene, La Jolla, California). pM primed using an appropriate primer according to the instructions in a). It was carried out using T1 petatar. Internal sequences were synthesized using synthetic primers (identified sequences It was obtained using a model prepared based on .

The complete nucleotide sequence and predicted amino acid sequence of MTI is shown in SEQ ID NO:1.

cDNA clones contain a polyadenylation signal (hypothetical start codon), a continuous open It has an open reading frame and codon usage consistent with human genes. MTI forecast The estimated amino acid sequence is 639 amino acids that encode a 70.5 kD protein with a PI of 5.47. It consists of amino acids. - The next structure is determined by the Chow-Fassman algorithm (C hou-Fasman, (1978) Adv, EnzyIllol.

Re1at, Areas Mo1. Biol, 47:145-148) As expected, 72% consisted of wax to alpha, 56% of which waxed to extension. It's a box.

The primary structure of MTI (presented in Figure 1) contains 27 proline residues, which are generally They appear in twos or threes throughout the molecule. The proline distribution within the sequence is As shown in Figure IA, the diamonds here represent proline residues. Proline pair and proline triblends are shown as stacked diamonds. 4 at the N-terminus A stretch of 0 amino acids contains a cluster of 8 prolines (residues 42-81) , which occur in pairs separated by three or fewer amino acids. Ru. A similar proline-rich region appears at the C-terminus (residues 551-563) of MTI. Thus, here 6 prolines appear within the 13 amino acid trench.

Both proline-rich regions have been described by Emini et al. (1985) J, ν1 ro1. ．． 55:836-839) based on the prohabili- ties If so, it probably exists on the fluorescent surface. This high proline neutrality also makes SDS polyacrylic Could you explain the unusual apparent molecular weight observed in lylamide gel electrophoresis? cormorant.

As mentioned above, the predicted molecular weight of MTI based on the amino acid sequence is 70.1 It is kD. However, as discussed below, naturally derived and &1IlI! ! The body protein migrates as a 90 kD protein on an SDS polyacrylamide gel. another As an option, this molecular weight variation can be achieved in both prokaryotic and eukaryotic cells. This may also be the result of post-translational modification.

Between the two proline-rich ends, the MTL lies in the region of an extended alpha-helinox structure. It has a matching sequence, which is shown by the hatched structure in Figure IB. Extension Helinox is A short helix, usually containing a pair of proline residues - is formed by a distorted amino acid stretch. 4 is obstructed in some places. Based on these theoretical calculations, predictions regarding the MTI structure can be made. A preliminary hypothesis is that this molecule is bounded by globular proline-rich domains at either end. It is said that it consists of elongated rod-like bodies that make up the .

Analysis of all available sequence databases has shown that MTI It was shown that it has a novel sequence that has no significant homology with Shiro. Furthermore, this The sequence of It seems that it does not have a motif.

Using cloned MTI DNA, total RNA from ME cells and polyA-containing R Northern blot analysis of NA was performed using standard methods on 15 μg of RNA. did. After performing the plot and hybridizing with Pi-identified pMTL DNA, Only one mRNA band was detected in the polyA-containing fraction. This band is open Tradiodaram was not detected in the total RNA fraction after 48-hour exposure; , indicating that MT mensage is a minor RNA species. northern bu Lonoto analysis suggests that MT1 protein is translated from a single mRNA .

Northern blot analysis also revealed that MTI RNA encodes the protein shown in SEQ ID NO: 1. 5" of the sequence. This upstream sequence contains one or more may represent further untranslated sequences and/or additional It may encode protein-coding sequences.

The MTI fusion protein was constructed using the insert from the pMT1 construct described above. No. 1 and the pMAL expression system from New England High Labs, Bihari, Masa. Obtained from Chusenotsu).

In this system, the gene in question (MT1) is kneed into the pMal-c vector. Rand Bio9f, Beverly, Massachusetts] and vector The protein in question and the maltose-binding protein were transfected into E. coli and expressed. We generated a fusion protein containing both. Maltose binding protein is the presence of maltose allows for selective purification of the fusion protein, followed by subsequent treatment with factor Xa. The complete recombinant MTI protein can be obtained by proteolytic cleavage. here Now, the start AUC codon is directly connected to the 5' end of the maltose binding protein. , MTlcDNA was cloned in pMAL-c vector. Fluorescence with factor Xa After degradative cleavage, the resulting MTL fusion protein has no -truncated additional amino acids; It retains the complete amino acid sequence encoded by MTlcDNA. pMAL system All experiments were performed in accordance with the manufacturer's instructions down to the last detail.

has an apparent molecular weight of approximately 90 kD and an outstanding electrophoretic mobility on 5DS-PAGE. have. Furthermore, the p+ values of both proteins are the same (5, 4), calculated from the amino acid sequences. Expected to be p ■ and lose. Leto & Marc hesi (1984) J, Biol, Chew, Qi4603-40 49), the cysteine residue was replaced with 2-nitro-5-thionanobenzoic acid ( In peptide mapping of both proteins cleaved with NTCB), Western plot Analysis yielded equivalent peptide fragments that shared the same MTI cross-reactivity.

Moreover, the number and size of the generated peptide fragments is similar to that of the proposed MTL amino acid. It was completely different from what was expected from the acid sequence.

Matata I Similar to MTI, MT2 expression levels were determined by serum and tissue culture supernatant analyses. significantly enhanced in various malignant cell types, as determined by both. below In the research described, the antibodies used were derived from two different cervical cancer cell lines (phylum E-180 and and Ca5ki, ATCC, Rockville, Maryland) for NMli products. It was created by

The 100 series antibodies were produced against ME-180, and the 300 series antibodies The second antibody was raised against the Ca5ki-NM immunogen. The following resistance In the body, 107.7 and 307.33 bind to MT2T2 protein and further 302-18.302-22 and 302-29 are closely related to MT2. It was found that this compound cross-reacted with proteins that were isolated together with it.

The results of the dose-response evaluation of the two antibody combinations are shown in Table II below. , ME-180 cell culture supernatant has been used as an antigen. Each annosé is an organization Demonstrates dose-dependent detection of antigen in culture supernatants, indicating that this assay is free from dead cells. We demonstrated that it is possible to quantify soluble inner nuclear matrix proteins undiluted. ．． 274 0.018 1:2 0.127 0.006 1:4 o, 067 0.006 1:8 0.035 0.009 1:16 0.021 0.007 No supernatant o, oo.

Upper aa average OD 5D 3ct o, 906 0.009 3:2 0.456 0.011 3:4 0.216 0.007 3:8 0.099 0.005 3:16 0.052 0.002 3:32 0.031 0.005 Next, internal nuclear matrix protein quantification experiments were performed on supernatants of various necrotic tumor tissues.

Here, tumors and normal tissues were killed in culture by serum starvation. In particular, cell lines were grown to contact with each other in tissue culture flasks using standard culture techniques. Continued The culture medium was replaced with serum-free medium, and the cells were incubated at 37°C with 5% CO2. It was kept in a container for 7 to 14 days. At the end of incubation, collect the culture solution and centrifuge it at 14,000Xg. The remaining cell layer was carefully removed. Investigated with Sandwich Inochianse with 11 compositions of supernatant. Ta.

The results are shown in Figure 3, where ME-180 antigen was used as a standard and all values is in units/g. As can be seen from Figure 3, MT2 antigen was released from each necrotic tissue. Increase in dead cells in tumor tissue when isolated and quantified as cancer tissue vs. normal tissue. reflected in higher MT2 mean antigen values.

Figure 4 shows a similar experiment performed using serum samples from cancer patients and normal blood donors. The experimental results are shown below. Here, the tissue is prepared as follows. remove tissue from donor Flash freeze in liquid nitrogen within 10 minutes to 4 hours after extraction. Store at -70°C until required. When ready for use, when the tissue is thawed, The tissue was aseptically cut into pieces of 0.1 to 0.3 cm squares in a hood with a fan. A serum-free culture medium containing Gyzon and gentamicin was placed in a separate flask. one Generally, 2-4 g of tissue is used in 100 ml of culture in a T150 flask. set The flask containing the fabric is then incubated at 37°C with 5% CO□ for 4-7 days. After keeping warm , the culture medium is collected from the flask and centrifuged at 4000×g for 20 minutes. Figure 3 The ME-18,0 cell antigen is ms. Results are given in units/ml, supernatant Control experiments in which the antigen is diluted in serum and the protein subsequently quantified in solution As can be seen from the results shown in Figure 4, (Similar to the results shown in Figure 3), serum samples from cancer patients differed from normal blood. compared to that detected in serum samples from cancer patients. Higher cell death as suggested by higher levels of MT2 antigen available reflects the speed of

cloning Following the same general methods as for MTI, selectively MT2-enriched compositions are ME- Obtained from 180 cells (cervical cancer cells, ATCC, Rock Pino Mutsu Maryland), M A T2-specific antibody was prepared.

107.7 antibody to screen the lambda ZAP expression library like MTI. A partial cDNA clone of MT2 was obtained by leaning. collect The resulting partial clone was then cloned into pBluescriptll hector (pMT 2) and sequenced the MT2 cDNA using standard techniques. Ta.

The sequenced DNA corresponding to residues 1366 to 2865 of SEQ ID NO: 3 is and analyzed to determine the reading frame and encoded amino acid sequence. complete The entire coding sequence was subsequently determined and is shown in SEQ ID NO: 3 (Compton et al. 1992) J, Ce1l Biol.

1i1395-1408), nucleotide sequence and predicted amino acid sequence of MT2 The acid sequence is set forth in SEQ ID NO:3.

The primary structure of MT2 is schematically shown in FIG. This protein is separated by a proline pair. (See Figures 4A and B) , this second-order structure allows the protein to form a coiled-coil structure in solution. do.

With respect to Figure 3, prolines are indicated by diamonds and helices are indicated by hatched boxes. difference Furthermore, both the C and N termini of MT2 appear to be folded like a globular domain. (Con+pton et al. (1992) J, Ce1l Biol, 11 nails 1395-1408).

The invention may be embodied in other specific forms without departing from its spirit or essential characteristics. It can be made concrete. The examples presented herein are therefore illustrative in all aspects. However, the scope of the invention is determined by the foregoing description, and is not intended to be limiting. is indicated by the appended claims and comes within the meaning and scope of the claims. Such modifications are included in the invention.

array list (1) General information: (i　i　i) Total number of arrays 26 (2) Information on array number 1 (D) Topology: linear (11) Molecule type: cDNA (F) Histological type: Cervical 11 ulcer (2) Information on sequence number 2・ (Xl) Molecule type: protein (x1) Sequence description Sequence number 2 Pro Glu Glu Val Ala Person 1a Arg Leu Ala Gin Gin (, lu Lys Glb Glu G1■ Vil Lys Ile ni lu Ser Leu Ala Lys Ser Leu　C1u　Asp　Ala　Leu　Arg　G1nSerG AiJGlyC1uLysLys5er人1aGasTrp人rgdinghrVa, 1C1uGly Person 1a Leu Lys Glu Arg Atg Lys Person la Van ALP Glu Ala Person Yua Asp AlaLeu L eu Lys ua Lys C1u Glu Ll! u Glu Lys M et Lys Ser Val X1e GluAI Ala Lys Lys Lys Glu Val Ala ni Lys Pro His Ileτhr AlaAla Glu Gly Lys Leu His A xn l1et Ile Val Asp LeuΔsp Asn Val V a1Lys Lys Val Gin da Ala Gh Ser Glu ua Lys Val Val Ser Gin TyrHis G11l L eu Val Val Gln Ala Arg Mu jp person sp Phe L ys person rg Glu Lau person 5■ Ser Ile Thr Pro Glu Van Leu Pro Gly Trp Lys Gly I (et Ser Val Ser Aap Leu A ia Asp Lys Leu Ser Thr Ajp jup Leums n Ser Leu Engineering 1e Alm Lys Ala Thr Giu Lys G lnHls Ileτhi Leu Aia Leu Glu Lys Gin LysVal Arg Person 5p Aha Met Glu Person sn Glu MeC Mrg Thr Pro Ser Pro Dingh: AlaLys Al a Asn Cys Serm sp mn Glu Fhe Thr Gin Ala　Leu　Thr　Ala　AlaGin　Ser　Leu　Leu　L eu Phe Pro Pro GlrIGin Leu Lys Pro P ro Pro to 1uTyr C'/s: le Glu His Gly h sp　Lsu　Glu　Leu　Ala　ua　Lys　Fhe　Val　As nGin LeuLys C1y Glu Se=＾rg Arg Val A la G′-nAspτ:p Leu Lys Glu (2) Information on SEQ ID NO: 3 : (11) Molecule type: DNA (B) Primary structure of heshi MA 4 (xi) Distribution IJ number 3 (2) Sequence number 40 information: (D) Topology: linear (11) Molecule type: protein (xi) Array description i! :Sequence number 4 Asp Glu Arg Ser Asn Arg Asp Glu Leu Glu　Leu　Glu　Leu　Ala　Glu　bnArg　Lys　Le a Leu Thr Glu Lys AJP Ala GLI rle Al a Met? let G button G-Arg Ile Asp hxg Leu person Yua Leu Leu Musn Glu Lys Gin Ala Person 1a S er　Pr.

Leu Glu Pro Lys Glu Leu Glu Glu Leu Arg Asp L7S Ain C1u Set Leu275 2g0 2 85 Thr Mec person = g Leu His Glu Thr Leu Lys Gin　C)rs　in　Asp　Leu　Lyi　τh■ Glu Lys Sir C; LI Met Asp Arg Lys Engineering 1e Person 5Ill Gin Leu Set Glu Glu ■Pn C1l)l A5P Leu Sir Phe Lys Leu human rg Gl u Phe Mla Ser Hls Leu Gin G attack■■ 325 3 0 3 5 C1y Lys Leu Se: [;in Leu Glu Glu [li s Leu Ser Gin Leu Gin jup A Tang■ Pro Pro Gin Glu Lys Gly Glu Val Leu Gly AJP Val Leu Gin Leu Gluτhr Leu L 7S CLI lu Ala Ala Tn: Leu Ala Ala A in Ain Thr Gin LauG Engineering n Muyu λ人rg val C1u Met Leu ni Thr Glu Arg Gly Gin Gin Glu 114] 5 440 445 Se = Ser Leu Ile Thr Asp Leu to Lo Ser Sir　IIs　Sir　hn　Leu　Ser　Gh+Thr　Ala　Gi n V&1Ala Ser Leu Thr Ser Glu Leu Thr Thr Leu Axn AlaGin Aln Leu Lys Glu Ser　Leu　Lys　Val　Thr　Lys　C1y　Ser　Leu　 Glu GluGlu Lys human rg Arg Ala muyu breath mus sp human Yua Lau Glu Glu Gin Gin rg Cys l1eAl a　Arg　Leu　Leu　Glx+　Leu　Gly　Glu　Ala　H ls G3s Ala C1u Thr to mu VaP 770 775 71!0 Thr to lu Lys Glu Gly Lys ASp [;in [;l u　Leu　Ala　Lys　Leu　Arg　L■■ Glu　Ala　Ala　Gin　Lys　Glu　Lsu　Glu Glu　Leu　Arg　Ginτhr　Vil　LysC1+lI　Leu　 Lys Glu Gin Leu Person 1a Lys Lys Glu Lys Glu) Hls person 1a Ssr fly Ser　Gly　Ala　Gin　Ser　Glu　Aua　Ala　C1y Arg Thr C1u Pro IThr Gly PrB Lys Leu Glu Ala Leu Arg Ala Glu Val Ser　Lys　Leu　Glu　Gin　l:ln　Cy■ Gly Gin Leu Glu GLu Lys Ala Gin Glu Leu Gly Hls Ser Gin Ser AlaLeu Ala S er　Ala　Gin^rg　Glu　Leu　Ala　Ala　Phe　Ar g Thr Lys Val G1nLeu Glu lu Ser Cys Ala Cys Cys Arg Gln Arg Gin Pro Person 1a Thr Va1Gin GLu Leu″’, = Ser Glr+ da Giu Arg Ala Glu Glu Leu Gly Gin Glu1 Ko L5 1320 1325 Gin Leu Ser Gin Lys Glu Gin Ala da G lu Hls T7r Lys Leu Gin Metλrg Ser ua Pro Muyua Ser Gin Ala Ser Leu Arg Person 1a Thr Ser　Ser　τhrLeu　Ser　Leu　! 'ra　C1y T7T Arg Pro Thr Thr Mu rg Sar Set Ala Arg Arg1B60 1865 11170 SerGinAlaGlyVtU SerSerGlyAlaProProGly ArgAjr1SerPheTF Met Gly Thr Cys Gin Asp Glu Pro Glu Gin Leu AspΔspτUmu5nmu rg Ile) in nu Leu GLrIGin Arg Jun A rg val Cys Pro Pro Hls LeuLys Thr Cy s Ty = Pro Leu C1u Sewmrg Pro Ssr Leu Ser Leu Gly Thr1925 1930 L935 Engineering 1e Thr A5P Glu Giu Met Lys Tbr Gly Human sp Pro Gln Glu Thr Leu Arghrg Ala S er Met in Pro Ile Gin tie Ala in mu ( , ly　Tbr　Gly　Ils　Tb■ Thr Arg Gin Gln Arg Lyx Arg val Ser Leu Glu Pro I (is (alb ni ly P Chichi B To Thr Pro Glu Set Lys Lys M11 Tbr Ser　Cys　! 'he Pro person rg Pro kl■■ τhr Pro Human rg Asp Arg His Glu G17 Arg Lyi Gin Ser Thr Thr Glu M1aGlrILys L ys Ala ua Pro Ala Ser Thr Lys Gin Al a　ALP　Arg　Arg　1nLeu　Leu　Arg　Arg　C,l y Aha Ser Lys Lys Iu & Leu Ser Lys AI JL Ser Ifr.

AJn Thr Arg Ser Gly Thr Arg ArgSer P ro　Arg　X1e　Ala　Thr　Thr　Thr人1a　Ser　Al h Ala Thr Ala person 1a mura Ile ni ly mu 1a Tb r Pro person rg person 1a Lys (2) Information on sequence number 5 (11) Molecule type: DNA (xi) Sequence description 7a: Sequence number 5: (2) Information on sequence number 6: (11) Molecule type: DNA (χI) Sequence description, SEQ ID NO: 6: 7χ Fu story πC YuωL Red πC^mouth Cυ discussion πCGC mouth Lτυ, τCC○, Gζso, C348FIG, 3.1 FIG. 3.2 FIG.4 average normal 3447 1284 5759 average cancer 9442 7069 26321 Submit a copy and translation of the written amendment (Patent Law Article 184-8) February 2, 1994, U]

Claims (1)

[Claims] 1. An isolated nucleic acid comprising a DNA sequence of SEQ ID NO: 1, including a variant thereof. 2. Hybridization with the DNA sequence of SEQ ID NO: 1 under strict hybridization conditions. Isolated nucleic acid to soybean. 3. A host cell transfected with the nucleic acid of claim 1 or 2. 4. A vector comprising the nucleic acid according to claim 1 or 2. 5. encoded by a DNA sequence containing variants thereof of SEQ ID NO: 1; A protein or protein fragment combined with a protein. 6. host cells by recombinant DNA having the sequence SEQ ID NO: 1, including variants thereof. A protein produced in a host cell and isolated from the host cell. 7. A binding protein that binds to an epitope on the protein of claim 6. 8. The conclusion of claim 7, wherein the binding protein is an antibody or an antibody fragment. Synthetic protein. 9. a) A set encoded by the DNA of SEQ ID NO: 1 or 3 containing a variant thereof Combining a recombinantly produced protein or protein fragment with an adjuvant to form a composition suitable for mammalian injection; b) The composition is injected into mammals to inject the recombinantly produced protein or protein block. inducing antibody production in the mammal against the fragment; c) an abnormality comprising steps a)-c) of isolating the antibody from the mammal; A method for producing antibodies used to detect different cell types. 10. The step of isolating the antibody from the mammal isolates the cells that produce the antibody. 10. The method of claim 9, wherein the method is carried out by isolating the coughing mammal from the coughing mammal. 11. (a) encoded by the DNA of SEQ ID NO: 1 or 3 or a variant thereof; A binding protein that recognizes an epitope on a marker protein containing an amino acid sequence (b) contacting the sample with the marker protein or fragment thereof; detecting the presence of a cell or cell nuclear residue, comprising steps (a) and (b); A method for detecting abnormal cell types in samples containing layers. 12. The method according to claim 9 or 11, wherein the abnormal cell type is a malignant cell type. . 13. The malignant cell type occurs in the bladder, breast, prostate, lung, colon, ovary or cervix. 13. The method of claim 12 having characteristics of a malignant cell type. 14. The binding protein binds to an epitope on the marker protein or protein fragment. 12. The method of claim 11, which is an antibody that specifically binds to a protein. 15. the antibody has a binding affinity of greater than 10 M-1 for the epitope; 15. The method of claim 14. 16. Claim 1, wherein the antibody has a binding affinity greater than 107M-1 Method in Section 5. 17. an additional step of quantifying the abundance of the marker protein in the sample. 12. The method of claim 11 comprising: 18. 12. The method of claim 11, wherein the sample comprises a body fluid. 19. If the body fluid concerned is serum, plasma, blood, urine, semen, vaginal secretions, cerebrospinal fluid, ascites, or peritoneal fluid, 19. The method of claim 18, wherein the method is selected from the group consisting of fluid, sputum, and breast exudates. 20. (a) encoded by the DNA of SEQ ID NO: 1 or 3 or a variant thereof; A binding that recognizes an epitope on a marker protein containing an amino acid sequence that indicates cell death. contacting the protein with the sample; (b) SEQ ID NO: 1 or 3 or a variant thereof released from cells of the tissue; The marker protein containing the amino acid sequence encoded by the DNA sequence or its a tissue comprising steps (a) and (b) of detecting the concentration of fragments of A method for determining the degree of cell death of a cell, the method comprising: detecting the marker protein or protein; The concentration of white fragments determines the degree of cell death, which is an indicator of the degree of cell death in the tissue concerned. How to determine. 21. c) increasing the concentration of the marker protein or protein fragment thereof at intervals; repeating the step of detecting; and d) an additional step of comparing the detected concentrations. 21. The method of claim 20, wherein the method comprises: 21. The method of claim 20, wherein the change is an indicator of the state of the tissue. 22. The decrease in the detected concentration becomes an indicator of a decrease in cell death, and the increase in the detected concentration Used to monitor changes in disease state or therapeutic effects, as an indicator of increased cell death. 21. The method according to claim 20. 23. If the tissue is breast, prostate, lung, colon, ovary, bladder or cervical tissue 21. The method of claim 20, which exhibits a characteristic. 24. a) mRNA transcription encoded by the DNA sequence of SEQ ID NO: 1 or 3 amino acid of SEQ ID NO: 1 or 3 or a variant thereof when translated. A sample with a nucleic acid that specifically hybridizes with the transcript of interest encoding the acid sequence. contact; furthermore, b) the mRNA transcript or fragment or variant thereof in the sample; A cell or cell nuclear remnant comprising steps a) and b) of detecting the presence of a species A method for detecting abnormal cell types in samples containing. 25. 25. The method of claim 24, wherein said abnormal cell type is a malignant cell type. 26. The malignant cell type is associated with breast, prostate, lung, colon, cervix, or bladder malignancy. 26. The method of claim 25 for characterizing cell types. 27. The nucleic acid is subjected to stringent hybridization conditions with the mRNA transcript. 25. The method of claim 24, wherein the method is hybridized with: 28. Claims that include an additional step of quantifying the abundance of the transcript in the sample. The method of scope item 24. 29. MT1 or MT2 mRNA transcripts or protein products, including variants thereof Use of molecules capable of binding with for the production of cancer therapeutic agents. 30. The cancer therapeutic agent is used for breast cancer, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, and colon cancer. or the use of claim 29 for lung cancer. . 31. The molecule is compatible with at least a portion of the DNA sequence of SEQ ID NO: 1 or 3. Use of claim 30 which is a complementary oligonucleotide. 32. The oligonucleotide is a synthetic oligonucleotide and has SEQ ID NO: 5 or 32. The use of claim 31, wherein: comprises at least a portion of the sequence 6. 33. the molecule substantially irreversibly interacts with MT1 or MT2 or a variant thereof. The use of claim 29 which is a member of a binding pair capable of binding. 34. The member of the binding pair has about 1 34. Use of claim 33 which binds with greater affinity than 09M-1. 35. Synthetic oligonucleotides mixed with pharmaceutical carriers used in the manufacture of therapeutic drugs. and the synthetic oligonucleotide is MT1 or MT2 or a variant thereof. The pharmaceutical carrier comprises a sequence complementary to at least a portion of the mRNA transcript of Synthetic oligonucleotides mixed with the body. 36. Compatible with at least a part of the DNA sequence of SEQ ID NO: 1 or 3 or a variant thereof 36. The synthetic oligonucleotide of claim 35, comprising complementary sequences. 37. comprising at least a portion of the sequence of SEQ ID NO: 5 or 6 or a variant thereof; The synthetic oligonucleotide of claim 35. 38. The synthetic oligonucleotide of claim 35 is at least 15 nucleotides in length. Ligonucleotide. 39. A binding protein used in the manufacture of a pharmaceutical, wherein the binding protein has SEQ ID NO. or 3 or a variant thereof, for a protein encoded by the DNA of about 109 A binding protein used in the production of the medicament, which has a binding affinity greater than M-1. .